Project Summary/Abstract Two to five percent of children born in the United States are afflicted with some form of Fetal Alcohol Spectrum Disorders (FASD). FASD causes numerous defects in affected individuals, commonly including facial phenotypes. These facial structures are derived from the neural crest, which appear particularly susceptible to ethanol teratogenesis. While the mechanism of this susceptibility is largely unknown, there is a genetic component. My host lab has performed several genetic screens to identify mutations which modulate ethanol sensitivity in craniofacial development. One mutation uncovered in these screens is in a novel gene I named lrp13b. This gene is a member of the low-density lipoprotein receptor related protein (LRP) family. My subsequent analyses have shown mutation of a second LRP gene, lrp2b, also causes ethanol sensitivity. This suggests LRPs may broadly mediate ethanol teratogenesis. LRPs act as receptors or co-receptors for over 50 ligands, including ligands involved in several ethanol-sensitive pathways. LRPs act on these pathways by promoting receptor-mediated endocytosis. Receptor-mediated endocytosis is known to be disrupted by ethanol exposure in the liver. It is possible ethanol exposure disrupts this process in the developing embryo. I hypothesize that ethanol teratogenesis is due to disruption of receptor-mediated endocytosis in genetically susceptible individuals. The first aim of this proposal will characterize the role of lrp13b and lrp2b in craniofacial development and ethanol sensitivity. I will characterize zebrafish mutant for these genes by observing the behavior of their neural crest. I will examine these cells? migration, proliferation, differentiation, polarization, and apoptosis. These experiments will uncover how loss of these genes leads to developmental ethanol sensitivity. In the second aim, I will determine the signaling pathways regulated by Lrp13b and Lrp2b. I will determine how loss of these receptors impacts the transcriptional targets of four developmental signaling pathways. I then examine genetic interactions between these LRPs and their suspected ligands. These experiments will determine the mechanism through which these genes impact development and ethanol sensitivity. In the final aim, I will determine the impact of ethanol exposure on receptor-mediated endocytosis in developing embryos. I will generate fluorescent labels of endocytic pits and observe their behavior in live embryos. I will then determine whether this behavior is disrupted by ethanol exposure. Next, I will determine whether disruption of endocytosis is sufficient to mimic ethanol exposure during development. I will use CRISPR-Cas9 to generate mutations in genes required for receptor-mediated endocytosis. I will then test these mutants for ethanol sensitivity. I will also determine whether loss of these genes can mimic ethanol exposure in ethanol-sensitive backgrounds. This aim will determine both whether ethanol disrupts endocytosis during development and whether that disruption is sufficient to cause FASD phenotypes. This proposal will provide unique insights to the mechanisms underlying genetic susceptibility to ethanol exposure and how ethanol disrupts cell behaviors.